FI106128B - Modified CD44 surface proteins, DNA sequences encoding them, antibodies recognizing and reacting with these proteins, and their use in diagnostics - Google Patents

Abstract

The inventon relates to antibodies that react with a variant epilope in the extracellular region of a variant CD44 polypeptide, wherein the variant epitope has the amino acid sequence: I S S T I S T T P R A P D H T K Q N Q D W T Q W N P S H S N P E V L L Q T T T R M T D V D R N G T T A Y E G N W N P E A H P P L I H H E H H E E E E T P H S T S T I O A T P S S T T E E T A T Q K E Q W F G N R W H E G Y R Q T P R E D S H S T T G T A A A S A H T S H P M Q G R T T P S P E D S S W T D F F N P I S H P M G R G H Q A G R R (residues 53-219 of SEQ ID NO:4). Methods of using the antibodies to identify variant epitopes also are provided.

Description

1 106128

Modified CD44 surface proteins, DNA sequences encoding them, antibodies recognizing and reacting with these proteins, and their use in diagnostics Varianta CD44 core protein, DNA sequencer, anti-ripple, identifier and reactor med dessa protein. deras användning i diagnostik

The present invention relates to modified CD44 surface proteins, antibodies which recognize and react with the modified determinants of these proteins and methods for their preparation, further to DNA sequences encoding these modified protein fragments, and to the use of these proteins or portions thereof or antibodies acting against them. in the diagnosis of metastases.

The very life-threatening nature of malignant tumor cells is due to their ability to form metastases. Original primary tumor cells acquire this property probably as a result of numerous changes during tumor development. As a result of these events, the cancerous cell variants which penetrate through the extracellular matrix and migrate continuously from the original tumor mass. lymphatic system or bloodstream. These metastasis-forming, often adherent, cancer cells move ··· 'into the circulatory or lymphatic system and exit the vein system at other sites where they invade other tissues to form lymphoid tumors (overviews / publications: Hart et al., 1989; Nicolson, 1987). The formation of metastases requires numerous interactions between the tumor cells and the intercellular matrix or other cells. Nearly - :·. all of these interactions require cell surface components such as receptors for the matrix and laminate, surface-bound proteolytic enzymes, and * 'cell adhesion molecules, including those that cause organ-specific adhesion and thus: that metastases are more susceptible to certain organs, in addition to growth factors and growth factor receptors.

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It is known that the membrane proteins of metastatic and non-metastatic tumor cells derived from BSp73 tumor in rats differ from each other as demonstrated by the antibody response (Matzku et al., 1983 and 1989).

Subsequently, it has been found that metastatic BSp73ASML tumor cells contain a surface protein that corresponds in part to a known glycoprotein involved in lymphocyte adhesion and cell-cell and cell-matrix interaction (the human normal glycoprotein, Ppg-1 and rat, respectively (HEBF1n). However, this novel abnormal or modified CD44 surface protein differs from these known sequences by a 154 amino acid extracellular domain (SUA), which is located between amino acids 220 and 237 in the human CD44 sequence (amino acids 224 and 239, respectively). between). This novel glycoprotein appears to be very important for cell matrix or cell-cell binding in the formation of metastases. Thus, the preparation of this protein region (SUA) and the elucidation of its characteristic features are one of the tasks of the present invention. Spleen cells that make up the transformed «t '; Antibodies directed against the SUA of CD44 surface protein were produced by immunizing mice with membrane-derived membrane-derived proteins from BSp73ASML. They were fused to myeloma cells according to Köhler's method (1981) using polyethylene glycol to obtain stable cultures. Antibodies that specifically target this novel protein moiety SUA are obtained by cloning and selecting cultures that produce antibodies reacting with BSp73ASML but not reacting with the basic non-metastatic form BSp73AS. nor do other non-neoplastic rat cells. For other studies, a monoclonal "*" * antibody was selected that stains BSp73ASML cells particularly strongly. . ·. ti in the immunofluorescence assay, and labeled as *. mAbl. 1ASML (mAb = monoclonal antibody).

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In the Westernblot assay, protein hydrolysis of BSp73ASML cells can be assayed for mAbl. 1ASML had 4 protein bands with molecular weights of 120,000, 150,000, 180,000, and 200,000, while extracts of rat fibroblast cells and rat non-metastatic tumor cells did not produce a significant reaction. It has not yet been possible to determine whether these size differences are due to different original amino acid sequences or to post-protein modifications of varying intensity. In any case, the antibody-recognized epitope is present in all 4 protein species, in the absence of comparison with proteins derived from non-metastatic BSp73AS cells or normal rat cells.

Isolation of cDNA sequences encoding surface protein SUA

Monoclonal antibody mAbl. 1ASML was used to find cDNA sequences encoding SUA in a bacterial expression library. This library was constructed using polyA + RNA derived from BSp73ASML cells and the pEX vector system (Stanley & Luzio, 1984). These cDNA sequences ... the products encoded are expressed as β-galactosidase fusion protein • * '; nasal antibodies. The cDNA clone positive for monoclonal antibody I1.11.11, thus designated pEX34, comprises a 167 nucleotide cDNA sequence fragment. This cDNA fragment was then utilized to examine the larger cDNA library present in vector pSP65, also obtained from::: BSp73ASML RNA. One of these clones isolated in this way, pM66, was then used, so-called "plaque" extension (starting oligonucleotide) and polymerase chain reaction (PCR), to aid in isolation of the full-length cDNA clone pMeta-1. Full length detection was obtained by primer extension '(3207 nucleotides). Collinearity BSp73ASML Tumor 9; mRNA was detected by RNase and SI · · · · · ·, protective analysis.

4,106,128 Isolation of cDNA sequences by bacterial expression and antibody recognition indicates that the antibody recognizes a primary amino acid sequence belonging to the surface protein SUA of the primary amino acid sequence.

The messenger RNA encoding SUA is expressed in BSp73ASML cells, but not in BSp73AS cells cDNA clones were generated three different sequence probes to detect specific messenger RNA molecules by hybridization. Probe A covers the cDNA region encoded by SUA (positions 941-1108). Probe B represents sequences between positions 1403 to 1572 and probe C comprises sequences extending from pMeta-1 to position 794 (Figure 1). The poly A + RNA of BSp73 tumor cell lines was electrophoretically separated and analyzed by RNA transfer hybridization. Four of the cell lines that do not form metastases do not contain RNA homologous to probe A, while the RNA molecules derived from the metastatic tumor cells BSp73ASML react strongly. Probe A recognizes from this RNA preparation a heterogeneous mixture of RNA molecules of various sizes in the range of 2.2-3.3 kb and one larger species of 4.9 kb RNA. The expression of specific membrane proteins recognized by monoclonal antibody 1. 1ASML exclusively in metastatic tumor cell variants apparently results from the specific expression of the corresponding messenger RNAs encoding SUA. Apparently a complete cDNA clone, pMeta-1,

The size of 3.2 kb cannot represent all sequences of this RNA species. It can represent only one species from a heterogeneous mixture of RNA molecules of different sizes. The same hybridization pattern as that of probe A is obtained for probes III 'B and C

4 '. In the resolution of BSp73ASML RNA, at least to the extent that can be stated within this heterogeneity, in other words, these RNA species comprise sequences that complement

:: *: all three probes A, B and C. Unlike probe A

• · <Probe B and C also recognize messenger RNA species in metastatic

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f i 5 106128 boiled in non-formed cell lines. However, these RNA sizes are clearly different from the lengths present in BSp73ASML cells, as four distinct messenger RNA species can be detected with 1.5, 2.0, 2.9, and 4.3 kb messenger RNAs. Although these RNA species may be hidden in a heterogeneous mixture of RNA molecules derived from BSp73ASML cells, it is certain that they will not be present in the same amount in BSp73ASML cells. Crucially, RNA sequences complementing probe A appear to be completely absent from cells that do not form metastases. Thus, it may be conservatively concluded that the sequences of probes A, B, and C are present in the same RNA molecules in the case of metastatic tumor, as if the sequences of probe A had been extended to BC positive RNAs, as if this alternative had occurred only forming a cell line.

S1 nuclease and RNase protection assays were performed to demonstrate collinearity between RNA and cDNA and to analyze the difference between RNA molecules derived from BSp73AS and BSp73ASML cells. Protected DNA or RNA fragments can only be shorter than the full length because the 5 'ends contain vector sequences that are unable to hybridize with either RNA molecules derived from plants. . First Ύ \ "look at the 3 'side pressure transitions: the transition sekvensseis- • · s having homology with the probe A, the sequences m · * * · ·· having homology with the probe B. Both technologies *. * a single RNA species is found in BSp73AS cells, which is * · «V * 'co-linear with a wide range of probes. In the 5 'direction, the cDNA or RNA probe corresponding to the RNA sequences obtained from BSp73ASML cells differs from that derived from BSp73AS cells. only RNA. RNA molecules derived from BSp73ASML cells. The liters primarily contain sequences that protect larger fragments of the probes. The largest chunks correspond to the full length of DNA or RNA fragments provided for screening analysis. Smaller pieces can also be shown. Since RNA transfer hybridizations have revealed a heterogeneous mixture of RNA molecules of various sizes, it is possible that these smaller protected fragments show RNA species that differ from cDNA at some other site, i.e., sites located at a previously identified point of departure and between the 5 'ends of the probes provided. Also, these RNA species cannot be detected in BSp73AS cells.

Next, we consider the 5 'flanking point, i.e. the transition from sequences hybridizing with probe C to sequences that hybridize with the coding sequence of probe A, SUA. Analysis yields similar results for the 5 'breakpoint. RNAs derived from BSp73AS cells can protect the provided probes in only a small region. Longer pieces are protected by messenger RNAs derived from BSp73ASML cells. Namely, they are collinear over the entire length of the probes provided. Thus, it can be concluded that the cDNA clone pMeta-1 represents sequences that are expressed in metastatic tumor cells BSp73ASML. The 3 'and 5' regions are also found in RNA molecules derived from BSp73AS cells. The SUA coding sequences whose encompassing transition sites can be mapped by these techniques described above indicate that an alternative extension mechanism must be present in RNA formation.

; The 5 'and 3' breakpoints of the transition sites leading to the SUA sequences are shown in Figure 1 with arrows.

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Monoclonal antibody 1. 1ASML recognizes a modified form of the glycoprotein • · · i.i: CD44 *: To obtain structural information from the surface protein, the sequence of all cloned cDNAs has been determined. The nucleotide sequence of the full-length; * ·, · clone pMeta-1 and its deduced amino- • «; acid sequences are shown in Figure 2. The full-length cDNA-4 'clone comprises 3207 nucleotides. The 3 'terminal has a polyA terminus, with two additional polyadenylation signals located at positions * 2288 and 1743. The first ATG codon is located downstream of the consensus start sequence and opens a reading frame of 1509 nucleotides corresponding to 503 amino acids. As expected for the membrane protein * »* f f 7 106128, the first 21 amino acids are hydrophobic and represent a signal peptide. To date, no part of these sequences has been found in the databases. However, within the scope of the invention, sequence homology was observed compared to recently published data on the Homing receptor lymphocytes CD44 (or Pgp-1, respectively) (Idzerda et al., 1989; Goldstein et al., 1989; Stamenkovic et al., 1989; Nottenburg et al., 1989; Zhou et al., 1989). These homologies are strictly limited to the 5 'and 3' portions of the cDNA, including the unreaded regions, and end at the above-mentioned divergence points between the BSp73AS and BSp73ASML RNA sequences. The entire breakpoint (indicated by color coding in Fig. 2), that is, the entire fragment encoding the SUA specific for metastases, is not present in the Pgp1 or CD44 sequences. Apparently, the colony specific glycoprotein is a variant of the CD44 glycoprotein. Namely, it comprises an additional extracellular domain of 156 amino acids and thus an expanded extracellular domain of 410 amino acids (minus the 21 amino acid signal peptide) as compared to the unchanged CD44 glycoprotein of 270 amino acids (also reduced by the signal peptide). However, non-colonizing BSp73AS cells contain unmodified forms of this CD44 family. The cDNA sequences of these BSp73AS RNA molecules have also been cloned, and similarity to metastasis-specific clones outside the extra domain.

There is a correlation between expression of CD4 4 variant and the ability to form metastases -V * In the context of the invention, rat isogenic tumors were studied. a second series of lineages, namely, tumor cell lines of system 13762 NF of the mammary carcinoma system (Neri et al., 1982), which sought to determine whether the altered CD44-gly-? * · * F 'co-proteins without exception in BSp73ASML cells and are related to the ability of these cells to form metastases or more generally metastasis formation. In this study, parental 9 1 * * β 106128 cell lines derived from the lymph node, i.e., MTPa, MTC, MTF7, and MTA (Group 1), were compared with lymph node or lung metastases, i.e., MTLγ, MTLn2, MTLn3 ( group 2). Group 1 cells express a substantially normal CD44 pattern, similar to RNA molecules derived from BSp73AS cells, when hybridized with probe B. In contrast, probe A detects a small amount of mixed RNA having a size of about 2.5 kb. On the other hand, Group 2 cells have a completely different RNA pattern. Both probes A and B hybridize to larger RNA species. These sizes are similar to the sizes indicated for BSp73ASML cells. This similarity is also confirmed by RNase and S1 protection assays. Based on these results, it is concluded that alteration of the RNA extension pattern and expression of the CD44 variant is in some way related to metastasis formation, and that the resulting pattern in these metastatic mammary carcinoma cells closely matches that described above for the metastatic BSp73ASML cell line. The high-molecular, antibody-identified proteins correspond to each of the high-molecular species identified in the BSp73ASML extracts. Hereby, the mammary gland tumor series states: v. thus, similarly to colony-specific expression of RNA species and large molecule proteins. The discovery of RNA that hybridizes to the coding probe A, or SUA, is found in Group I, the so-called parent cell lines. , and that a slight staining of a protein with a molecular weight of "100,000 Daltons can be detected by the antibody, · · · * is considered to be due to the fact that Group 1 cells also have little ability to form far-flanking cells. than the original tumor cell line: * · *: BSp73AS, which had no behavior leading to metastasis.

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Monoclonal Antibody 1.1ASML Recognizes the Formation of Metastases in Rat • · • «• In a series of experiments to form metastases on tumor cells of 106108 lineage BSp73ASML in rats, these cells were injected subcutaneously, and at two different times, the monoclonal antibody 1.1ASML ... every three days before and after the administration of the tumor cells. This immunoassay also determined how the rat immune response to the injected antibody developed. Namely, the animals developed anti-mouse immunoglobulin antibodies and anti-idiotype antibodies. As a result of this series of experiments, 1.1ASML injections significantly retarded tumor adhesion and metastasis. The kinetics of this delay can be inferred that the antibody interferes with some of the primary processes involved in metastasis formation. This experiment demonstrates that the antibody-recognized protein structure located on the surface of the metastatic cells plays some role in the formation of the metastases and the diagnostic objectives are realistic.

Isolation of a human sequence homologous to the SUA coding sequence of rat cDNA

Of course, in the case of cultured human derived tumor cells, there is no obvious

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• as in the rat system, whether the cells still have metastatic properties. Based on tests performed in immunocompromised mice *. * can only draw very limited conclusions about the ability to · · ·: form metastases in humans. For this reason, the relatively large number of tumor cell lines cultured some time ago in some part of the world had to be examined for the presence of sequences that could be demonstrated as follows. rat metastases within the scope of the invention. The frame of the invention you managed to find one such tumor cell line.

It is derived from human large cell lung cancer and has been designated LCLC97. From this tumor cell line three specific RNA species (sizes: 5.5, 3.4, and 2.8 kb) that behaved completely like the 10 106128 RNA molecules that could have been detected in rat metastatic tumor cell lines, of which. Namely, they hybridize to probe A as well as probes B and C, which means that these human RNA species are also broadly similar to the cDNA pMeta-1 clone (85%).

However, the monoclonal antibody 1.1ASML did not react with this tumor cell, i.e., the protein portion recognized by the antibody must distinguish, in terms of the antigenic determinants region, from the proteins present on the surface of the human tumor cell. Even small deviations due to the high specificity of the antibodies are sufficient for non-response. This human tumor cell LCLC97 was now used solely to construct a cDNA library. Based on the high degree of concordance between rat and human sequences, one cDNA clone having homology to probe A could be isolated. The sequence of the human cDNA was determined. Figure 3 (a, b) shows the primary chain and the deduced amino acid sequence. It can be noted that rat and human sequences are similar over large areas. This human sequence as well as the deduced amino acid sequence are likewise disclosed in the appended patent. screaming.

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Execution Examples • Cells and Antibodies · · · · · · ·

The following cloned Bsp cell lines were used for the studies: BSp73 14ASML-1 and 10AS-7 maintained in cultures; as described in Matzku et al., (1983); in addition, the cell lines for mammary gland cancer described in Neri et al., (1982).

The monoclonal antibodies recognizing BSp73 ASML membrane proteins were made by immunizing Balb / c mice. After the spleen cells from the immunized mouse were isolated, they were fused to Ag8 myeloma cells to obtain ..... 106128 into the tomato form using the method of Köhler (1981) for the production of monoclonal antibodies. The hybridoma cells obtained in this way were screened for cells which produced a cell specific antibody against BSp73ASML cells. however, against BSp73AS cells and rat normal fibroblast cells The exact procedure is described in Matzku et al. (1989).

As desired, hybridoma cells producing specific monoclonal antibody (mAk) were allowed to proliferate in tissue culture and mAk secreted into the culture medium was concentrated by heavy precipitation with ammonium sulfate and assayed by column chromatography (Protein A-Sepharose). One of these antibodies is mAkl. 1ASML.

Immune fluorescence

To detect the modified CD44 molecule on the surface of various tumor cells, these cells were transferred to culture, washed with phosphate-buffered saline (PBS) and incubated with 1.1ASML for 30 minutes at 40 ° C. Rhodamine-coupled rabbit anti-mouse IgG was used as a secondary antibody to detect binding and was observed under a fluorescence microscope.

Construction of Immuno Screening and Immuno Screening of cDNA Expression Libraries Oligo (dT) and various hexa-PP · nucleotides were used as a primer for polyA + RNA from BSp73ASML cells, and The first strand of cDNA was synthesized. . With the help of the reverse transcription enzyme from AMV. The second strand of cDNA was prepared by E. coli DNA polymerase I RNase H and 'E. coli ligase, after which the ends of the double-stranded cDNA: ··: were repaired with T4 DNA polymerase. Vectors pEX1, 2 and 3 (Stanley and Luzio, 1984), which allow fusion of cDNA into three different reading frames, were opened.

SmaI restriction endonuclease and ligated with cDNA "10612812 (T4 DNA ligase). Capable E. coli DH5 bacteria (pCI857) that produce a temperature-sensitive repressor are transfected with pEX cDNA and cultured on nylon filters. The temperature-sensitive repressor RCI857 gene is located in plasmid pCI857, which is compatible with pEX plasmids. The IPR promoter, which directs fusion protein synthesis, does not function at 28 ° C. When the temperature is raised to 42 ° C, the CI repressor is inactivated and the synthesis of β-galactosidase / ASML fusion proteins becomes very potent. The heat-induced bacterial colonies on the filters are then denatured with chloroform vapor, then incubated in PBS containing 3% dry milk powder, lysozyme and DNase. The bacterial fusion proteins attached to these nylon filters are now incubated with mAkl. With 1ASML, the nonspecifically bound mAk 'is washed away, followed by binding using iasI-labeled rabbit anti-mouse IgG as a secondary antibody. Following auto-radiography, positive clones were isolated from the original bacterial filter and analyzed in their outline. One clone that fusion protein synthesized specifically with 1.1ASML was pEX34. The pEX plasmid contained in the bacterial clone comprises a 167 nucleotide cDNA encoded by e.g. an epitope (or antigenic determinant) with which mAkl. 1ASML is specific.

Ill ...: Isolation of full-length cDNA mMeta-1 was then performed according to standard standard procedures.

• Immunization of rats mAkl. 1ASML was injected subcutaneously or intraperitoneally in BDX rats syngeneic with BSp73 tumor cells mAkl. 1ASML (attached to keyhole limpet mollusc hemocyanin) together with Freund's complete excipient. The first injection was given (to the paw fat layer) 10, 7 and 3 days before the injection of BSpASML cells, the following «. . ·. injections were then given at 3, 7, 11, 14 and 21 days • i ·. ···. after this. Rats are sacrificed after 28 days, lymph nodes are separated, weighed and macroscopically visible metastases in the lungs are counted.

Relationship between expression of altered CD44 surface proteins and metastatic capacity

A second set of rat tumor lines obtained from 13762NF mammary carcinoma (Neri et al., 1982) sought to determine whether expression of altered CD44 glycoproteins is merely a feature of the studied BSp73ASML cell line, or whether it can be expressed. In addition, cell lines derived from primary tumors [MTPa, MTC, MTF7 and MTA (group 1)] were compared with cell lines derived from lymph node and lung metastases [MTLy, MTLn2, MTLn3 (group 2)]. The pattern of RNA from CD44 is shown in Figure 4, with probes A, B and D corresponding to page 4 of this application and with the probes described in Figure 1. All Group 1 cells produce a normal CD44 pattern using probe B. In contrast, group 2 cells provide a pattern different from this. RNA is larger than Group 1 RNA and corresponds to BSp73ASML RNA. Smaller RNAs are lost upon probe D hybridization. The remainder of the figure shows similarity between the rat, both tumor systems.

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Also, the group 2 RNA pattern obtained using probe A corresponds to the pattern of BSp73ASML. Probe A does not hybridize to RNA from BSp73ASML 99, whereas group 1 does not hybridize to: ·; the bones provide a small confused 2.5 kb RNA band. The protection assays performed with RNase and S1 nuclease also show structural similarity. Based on these results: a change in the pattern and expression of the altered CD44 RNA molecules occurs with the formation of metastases.

«» 4 '"· Transferring Remote Colonization Capacity by Overexpressing pMeta-1 in Non-Colonizing BSp73AS Cell • · s • · 14 106128

The relationship between expression of altered CD44 species and metastatic capacity in two rat tumor series suggests that these glycoproteins have a causal relationship to metastasis formation. To investigate this, pMeta-1 was transferred to BSp73AS cells, and then examined whether this alters the behavior of these cells. The complete coding region of pMeta-1 (Figure 2) was placed under the SV40 promoter, and this construct (Figure 5 in Figure 5) was co-seeded with BSV73AS into PSp2AS. In this way, individual colonies resistant to G418 and expressing pMe-ta-1 were obtained. Figure 5 shows an RNA pattern obtained from two colonies. Hybridization of probe A specific for the modified CD44 shows a predominant copy of about 2.2 kb, which corresponds in size to the smallest common RNA copied in BSp73ASML cells (Figure 5). However, transformed cells contain this RNA approximately 10-fold more than BSp73ASML.

Other orders of magnitude are found in one transfected cell (BSp73ASML-pSVMetal-14), which may depend on the plasmid integration site. The pSV2neo simulation transfer clone (not shown) and the receiving BSp73AS cells do not contain RNA that would be a complement to probe A. To detect endogenous normal CD44 copies (without the additional: domain of pSVMeta-1) in the transfectants, the filter was exposed and hybridized to probe D. This portion of the unreaded · · · 3 'sequence is not present in the s clone (cf. j Figure 5). Probe D shows two major copies of 2.9 and 4.9 · * · · · kb in each transfected ant n RNA (Fig. • · 5 right column), both in comparison with BSp73AS and also BSp73AS-pSVneo: not shown.

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Approximate quantification of various compatible hybrid mutants indicates that these transfectants express about 5 * * times more modified CD44 RNA copied by the expression plasmid than endogenous gene copies.

• · * • · * • · · * * • · · | "106128

Overexpressed cDNA is considered a protein. Each transfectant shown in Figure 5 synthesizes mAbl. 1ASML makes immune-staining proteins seemingly the same size, namely the 150 kDa major product and the 100 kDa weaker band. Because this cDNA sequence encodes a primary protein product of only 503 amino acids (corresponding to about 60,000 Daltons), all visible bands must present modified forms. The 150 kDa band is consistent with a modified form of the modified CD44, which is expressed in the metastatic cell BSp73ASML. BSp73AS or simulated transplanted BSp73ASpSVneo cells do not comprise this protein. As with BSp73ASML cells, the epitope of cells expressed by transfectants is exposed on the cell surface.

To demonstrate that the expression of altered CD44 is sufficient to confer metastatic capacity on BSp73AS cells, transfectants were injected into syngeneic BDX rats (spontaneous metastasis method). In previous experiments, metastatic tumor cells BSp73ASML spread rapidly from the injection site and were completely dispersed about 10 days after injection (Matzku, 1984). All local tumors were therefore removed by dissection on day 10. All lungs of carriers of BSp73ASML cells and all animals injected with overexpressing transfectants developed metastases (Table 1). The metastasis of the metastases occurred relatively quickly, 5 to 8 weeks after injection. Animals that had received BSp73AS cells or simulant transfectants after this time were completely healthy (except for surgery), and no metastases were present: even after 5 months.

• · 1 · <* · · V · 4 \ In spite of the striking similarity observed in strong metastasis formation, "some interesting differences are also observed. BSp73ASML cells reach lymph nodes in all animals and induce marked enlargement of the Table 1.) The transfectant (BSp73AS-pSVMetal-14) causes lymph node enlargement in 3 out of 8 animals, although numerous metastases develop in the lungs of all animals (Table 1) .The second transfectant (BSp73AS-pSV-metal-15) does not detect a large lymph node. these transfectants appear to be able to form colonies in the lungs without the mandatory growth phase in lymph nodes.

The experiment of Table 1 further refers to another difference between BSp73AS transfectants and BSp73ASML cells. Individual lung metastases are macroscopically visible while BSp73ASML-induced metastases are small and numerous, however, in a larger series of experiments on BSp73ASML cells (Reber et al., 1990), 11 of 20 animals develop 5-20 larger colonies per lung. -fectants.

To verify that the resulting metastases are due to the injected transfectants, a spontaneous mutation that results in metastatic capacity, thus excluding, epitope-positive proteins were determined from whole lung extracts, and from the secondary culture. · ... from extracts from cells. The 150 kDa glycoprotein? · · Can be detected in whole lung extract as well as in specific lung colony extracts of animal that received BSpAS-pSVMetal-15 transfectants. The G418-resistant strain expresses a protein having this same molecular weight during in vitro growth.

Diagnostics 1. Human tumor material is analyzed by in situ hybridization to existing human pMeta-1 sequences.

. ·. : These experiments are intended to be pre-tests before obtaining an antibody that recognizes human SUA.

lv 106128 2. Production of human SUA-recognizing and reactive antibodies. Human pMeta-1 sequences are cloned into bacterial expression vectors to generate fusions of β-galactosidase or tryptophan E product. Rabbits are immunized with these fusion proteins or synthesized peptides (linked to carrier molecules) derived from SUA. Polyvalent or monospecific antibodies are isolated.

Uses: - Immunohistological examination (diagnostics) of clinical tumor material; - detection of soluble SUA in patient serum by ELISA assays (diagnostics); 3. preparation of hMeta-1 protein by transfecting human or rat cells with an expression vector comprising the complete hMeta-1 cDNA sequence; or purification from LCLC97 cells.

Uses: • Injection of a protein or portion thereof to identify and characterize binding sites for tumor cells · · · · · · · · · · · · ♦ · «• · • 106 106 106 12 12 12 12 12 12 106128

Bibliography:

Goldstein, L. A., Zhou, D. F. H., Picker, L. J., Minty, C. N., Bargatze, R. F., Ding, J. F. and Butcher, E. C. (1989) The human lymphocyte homing receptor, the Hermes antigen, is involved in the cartilage proteoglycan core and link Proteins. Cell 56: 1063-1072.

Hart, I. R., Goode, N. T. and Wilson, R. E. (1989) Molecular aspects of the metastatic cascade. Biochim. Biophys. Acta 989: 65-84.

Idzerda, R. L., Carter, W. G., Nottenburg, C., Wayner, E. A., Gallatin, W. M. and St. John, T. (1989) Isolation and DNA

sequence of a cDNA Clone encoding a lymphocyte adhesion receptor for high endothelium. Proc. Natl. Acad. Sci. U.S.A.

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Characterization of Bsp 73, a spontaneous rat tumor and its in. . vivo selected variant showing different metastatic capacities. Inv. Met. 3: 109-123.

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* / .: Neri, A., Welch, D., Kawaguchi, T. and Nicolson, G. L. (1982) * * * / <· '. Developmental and biological properties of malignant cell sublines * I <and clones of spontaneously metastatic rat mammary adenocarcinoma. J. Natl. Cancer Inst. 68: 507-517.

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Nicolson, G. L. (1987) Tumor cell instability, diversification, and progression to the metastatic phenotype: from oncogene to oncofetal expression. Cancer Res. 47: 1473-1487.

Nottenburg, C., Rees, G. and St. John, T. (1989) Isolation of mouse CD44 cDNA: structural features distinct from the primate cDNA. Proc. Natl. Acad. Sci. U. S. A. 86: 8521–8525.

Stamenkovic, I., Amiot, M., Pesando, J.M. and Seed, B. (1989) The lymphocyte molecule implicated in lymph node homing is a member of the cartilage linking protein family. Cell 56: 1057-1062.

Stanley, K. K. and Luzio, J. P. (1984), Construction of a new family of high efficiency bacterial expression vectors: identification of cDNA clones for coding for human liver proteins. EMBO J. 3: 1429-1434.

Wenzel, A. (1986) Characterization of Differenzierungs-antigenen auf dem Rattentumor Bsp 73 mit Hilfe Monoclonal Antibody. Master's thesis, University of Karlsruhe.

Zhou, D. F. H., Ding, J. F., Picker, L. F., Bargatze, R. F., Butcher, E. C. and Goeddel, D. V. (1989) Molecular Cloning and Expression of Pgp-1 - The Mouse Homolog of Human H-CAM.

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(Hermes) lymphocyte homing receptor. J. Immunol. 143: 3390-3395.

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table 1

Metastatic spread of BSp73AS cells expressing modified CD44 cDNA pMeta-1 ***

Local Dispersion in Finding Remote Tumor Colonies by Autopsy Clone_tyrnine_LN1 ing_LN1 par_Lung BSp73ASML 0/8 8/8 8/8 8/8 0 1, 5-2, 52 0 2.2-5.0 Granular BSp73AS- 0/8 3 / 8 3/8 8/8 pSVMetal-14 0 0.3-1.2 0 1.0-4.5 multiples.

0 0, 3-5, 0 BSp73AS- 0/8 0/8 0/8 8/8, 5-20 pSVMetal-15 0 0.3-10.0 BSp73AS 1/8 0/8 0/8 0/8 BSp73AS- 0/8 0/8 0/8 0/8 pSVneo * LN = lymph nodes ** Mean diameter in millimeters *** The table shows the condition 60 days after injection of said cells.

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Claims (14)

1. A DNA fragment, soin encodes a portion of a surface protein in metastasis-forming tumor cells, characterized in that this DNA fragment is selected from the group of a) nucleotide sequences ... rMeta-1 ... ATT GCA ACT ACT CCA TGG GTT TCT GCC CAC ACA AAA CAG AAC CAG GAA CGG ACC CAG TGG AAC CCG ATC CAT TCA AAC CCA GAA GTA CTA CTT CAG ACA ACC ACC AGG ATG ACT GAT ATA GAC AGA AAC AGC ACC AGT GCT CAT GGA GAA AAC TGG ACC CAG GAA CCA CAG CCT CCT TTC AAT AAC CAT GAG TAT CAG GAT GAA GAG GAG ACC CCA CAT GCT ACA AGC ACA ACC TGG GCA GAT CCT AAT AGC ACA ACA GAA GAA GCA GCT ACC CAG AAG GAG AAG TGG TTT GAG AAT GAA TGG CAG GGG AAG AAC CCA CCC ACC CCA AGT GAA GAC TCC CAT C-TG ACA GAA GGG ACA ACT - GCC TCA GCC CAC AAC • ·: '' AAC CAT CCA AGT CAA AGA AGT ACA ACA CAG AGT CAA GAG GAT GTT TCA TGG ACC GAT TTC TTC GAC CCA ATC TCA CAT CCA ATG <· «• * * '' GGA CAA« »*« * «'i'! and F* . . .hMeta-l. . . r «» i IC AAC CCA AGC CAT TCA AAT CCG GAA GTG CTA CTT CAG ACA ACC ACA AGG ATG CAT GAT GTA GAC AGA AAT GGC ACC ACT GCT TAT GAA GGA AAC TGG AAC CCA GAA GCA CAC CCT CCC CTC ATT CAC • · · "'CAT GAG CAT CAT GAG GAA GAA GAG ACC CCA CAT TCT ACA AGC I' · .. ACA ATC CAG GCA ACT CCT AGT ACA ACG GAA GAA ACA GCT ACC CAG AAG GAA CAG TGG TTT GGC AAC AGA TGG CAT GAG GGA • · · · TAT CGC CAA ACA CCC AGA GAA GAC TCC CAT TCG ACA ACA GGG • · · · ACA GCT GCA GCC TCA GCT CAT ACC AGC CAT CCA ATG CA in point (a), said DNA sequences and / or are their allelic variations. 106128 DNA pieces according to claim 1, characterized in that they contain nucleotide sequences which hybridize with one of the nucleotide sequences mentioned in claim 1a) and encode a complete surface glycoprotein in metastasis-forming tumor cells. The DNA fragment of claim 2, characterized in that it hybridizes to a nucleotide sequence as mentioned in claim 1, wherein the sequence of at least 85 percent is homologous to the reaction partner. A recombination DNA molecule, characterized in that it comprises a vector-based nucleotide sequence and a piece of DNA according to claims 1, 2 or 3. The recombination DNA molecule of claim 4, characterized in that it is an expression vector. * «« · 6. A transformed host cell, characterized in that it contains a DNA fragment according to claim 1, 2 or • 3: or a recombination DNA molecule according to claim 4 or III. I 3,> I «II 1 (1« «·« II 7. A polypeptide useful as a diagnostic agent, characterized in that a piece of DNA as claimed in claim 1 encodes it. »· •» A polypeptide used as a diagnostic agent, characterized in that it can be prepared by a recombination DNA molecule according to claim 4 or 5 comprising • · · ** ··. a piece of DNA according to claim 1. • · · · · · · · The polypeptide used as a diagnostic agent according to claim 7 or 8, characterized in that it contains the amino acid sequences! 106 128 R protein IATTPWVSAHTKQNQERTQWNP IHSNPEVL LQTTTRMTDIDRNSTSAHGENWTQEPQPPF NNHEYQDEEETPHATSTTWADPNSTTEEAA TQKEKWFENEWQGKNPPTPSEDSHVTEGTT ASAHMNKPSQRMTTQSQEDVSWTDFFDPIS HPMGQGKQTESK and h-protein ISSTISTTPRAFDHTKQNQDWTQWNPSHSN PEVLLQTTTRMTDVDRNGTTAYEGNWNPEA HPPLIHHEHHEEEETPHSTSTIQATPSSTT EETA-TQKEQWFGNRWHEGYRQTPREDSHST TGTAAASAHTSKPMQGRTTPSPEDSSWTDF / '; FNPISHPMGRGHQAGRR (4 <<4 and their allelic variations and glycosylation products. I I I 4 I Multiple or monovalent anti-protein antibodies used as diagnostic agents according to claim 7, characterized in that they have been prepared by using *** DNA according to claim 1 or proteins according to patent. ··· 'requirement 7. • · • 4 • · · Use of a piece of DNA according to claim 1, its • · · *. complementary / homologous complement strands, its derivatives, and its parts for identification, preparation or isolation of a nucleotide sequence, or its moieties, which encode a portion of a surface glycoprotein in metastasis-forming tumor cells. Use of an antibody according to claim 10 for identification, preparation or isolation of a polypeptide according to claims 7-9. 106128 A substance for the diagnosis of metastasis-forming tumors and / or tumor metastases, characterized in that it contains at least one antibody according to claim 10. The substance according to claim 13, characterized in that, an antibody is joined to an enzyme or marked with a dye and / or a radioisotope. p> I f (III • c II f • »f • I (lii I • tr» I («I« · <• I <«I · • · • · · • 1 · · • t • »• t« • · · · • · • · I • M • 9